Campylobacter jejuni is currently one of the most common bacterial causes of diarrheal disease in humans in the United States. It has also been strongly implicated as one of the most common infections to precede the development of Guillian-Barre' syndrome, an autoimmune disease. The study of C. jejuni pathogenesis has identified few potential virulence factors. One of these is the exotoxin, cytolethal distending toxin, (CDT). Recent work has identified that the C. jejuni CDT is a member of a new family of toxins that can cause a G2 cell cycle block in certain mammalian cultured cells via direct DNA damage and invocation of the DNA damage checkpoint pathway. However, little is known about the specific interactions of the CDT subunits, CdtA, CdtB, and CdtC, with sensitive cells. Nor is much known about CDT subunit interactions within the CDT holotoxin. This information will be crucial for gaining an understanding of how to best develop vaccine strategies and interventions in diseases caused by bacteria that produce CDT. The broad, long-term goals of this proposal are to understand how CDT interacts with sensitive cells. In particular, we will determine the nature of the Cdt subunit interactions with the surface of sensitive cells, and explore the nature and extent to which the Cdt subunits interact with each other. These studies are central to our understanding of how CDT affects mammalian cells. The specific aims of the proposal are to test the hypothesis that the individual Cdt proteins have specific capabilities involved in recognizing a specific mammalian cell surface receptor, and in interacting with each other to form an effective holotoxin. In Aim 1, a variety of mutations will be made in each of the Cdt subunits. The effects of the mutations on toxicity, holotoxin assembly, and cell binding will be tested. In Aim 2, two different approaches will be used to identify areas within the Cdt proteins that are responsible for mediating the association of the subunits into holotoxin. In Aim 3, we propose to characterize and identify the CDT cell surface receptor. These studies should substantially increase our knowledge of CDT biology and allow for the development of new treatment and prevention strategies.